This invention relates to polypeptides containing IgE sequences, especially IgE antagonists and to polypeptides capable of differential binding to Fc.epsilon.RI and Fc.epsilon.RII.
IgE is a member of the immunoglobulin family that mediates allergic responses such as asthma, food allergies, type 1 hypersensitivity and the familiar sinus inflammation suffered on a widespread basis. IgE is secreted by, and expressed on the surface of, B-cells. IgE synthesized by B-cells is anchored in the B-cell membrane by a transmembrane domain linked to the mature IgE sequence by a short membrane binding region. IgE also is bound to B-cells (and monocytes, eosinophils and platelets) through its Fc region to a low affinity IgE receptor (Fc.epsilon.RII, hereafter "FCEL"). Upon exposure of a mammal an allergen B-cells are clonally amplified which synthesize IgE that binds the allergen. This IgE in turn is released into the circulation by the B-cells where it is bound by B-cells (through the FCEL) and by mast cells and basophils through the so-called high affinity receptor (Fc.epsilon.RI, hereinafter "FCEH") found on the surface of the mast cells and basophils. Such mast cells and basophils are thereby sensitized for allergen. The next exposure to the allergen cross-links the Fc.epsilon.RII-bound IgE on these cells and thus activates their release of histamine and other factors which are responsible for clinical hypersensitivity and anaphylaxis.
The art has reported antibodies capable of binding to FCEL-bound IgE but not IgE located on FCEH (see for example WO 89/00138 and U.S. Pat. No. 4,940,782). These antibodies are disclosed to be clinically advantageous because they bind to IgE found on B-cells or circulating free in the body, but do not bind to FCEH and thus will not activate mast cells or basophils.
It is generally understood that FCEH, like FCEL, binds to a recognition site in the IgE constant (Fc) domain. The IgE recognition sites for the two receptors are poorly defined, despite considerable effort in the past directed to the problem.
Over the past decade several studies have been undertaken to determine which portion of the IgE molecule is involved in binding to Fc.epsilon.RI and Fc.epsilon.RII. Essentially three approaches have been tried. First, peptides corresponding to specific portions of IgE sequence have been used as either competitive inhibitors of IgE-receptor binding (Burt et al., Eur. J. Immun. 17:437-440 [1987]; Helm et al., Nature 331:180-183 [1988]; Helm et al., Proc. Natl. Acad. Sci. 86:9465-9469 [1989]; Vercelli et al., Nature 338:649-651 [1989]; Nio et al., Peptide Chemistry p203-208 [1990]) or to elicit anti-IgE antibodies which would block IgE-receptor interaction (Burt et al., Molec. Immun. 24:379-389 [1987]; Robertson et al., Molec. Immun. 25:103-113 [1988]; Baniyash et al., Molec. Immun. 25:705-711 [1988]). For competitive peptides, the best that could be achieved was a sequence that was 1000-fold less active than IgE (Burt et al., Eur. J. Immun. 17:437-440 [1987]).
Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 (1989) found that a peptide corresponding to residues 329-409 blocked in vivo sensitization of human basophil granulocytes with human IgE antibodies. Further studies indicated that residues 395-409 were not essential for binding of the 329-409 peptide to Fc.epsilon.RI (Helm et al., Proc. Natl. Acad Sci. 86:9465-9469 [1989]). Note that the IgE described below had the sequence of Padlan et al., Mol. Immun., 23:1063 (1986), but that the IgE residue numbers used herein are those of Kabat et al. Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. 1987).
Vercelli et al., Nature, 338:649-651 (1989) used recombinant IgE peptides as well as anti-Fc.epsilon. monoclonal antibodies to investigate the B-cell (Fc.epsilon.RII) binding site of human IgE. They concluded that the Fc.epsilon.RII binding site is in FC.epsilon.3 near K399-V402.
Burt et al., Eur. J. Immun., 17:437-440 (1987) investigated seven peptides for competition against rat IgE in binding to rat mast cells. Their most active peptide, p129, was 1000-fold less active than IgE. p129 corresponds to human sequence 439-453 which includes loop EF. Another of their peptides, p130, corresponding to residues 396-419 in the Fc.epsilon.3 domain, had no activity.
Robertson et al., Molec. Immun., 25:103-113 (1988) assessed IgE binding by sequence-directed antibodies induced by several synthetic peptides. They concluded that the sequence defined by their .epsilon.-peptide-4 (corresponding to residues 446-460), were not significantly involved in receptor binding while the sequence defined by their .epsilon.-peptide-3 (corresponding to residues 387-401), was likely to be proximal to the IgE-receptor recognition site.
Nio et al., Peptide Chemistry, p203-208 (1990) evaluated numerous peptides with respect to their ability to inhibit histamine release by human basophils in vitro. Only one peptide (peptide 2, Table 1), exhibited specific inhibition; this peptide encompassed residues 376-388. However, a larger peptide which incorporated this sequence (peptide 3, Table 1), had no inhibitory activity.
Second, mutations in IgE have been partially explored. Schwarzbaum et al., Eur. J. Immun., 19:1015-1023 [1989] (supra) found that a point mutant P404H (P442H herein) had 2-fold reduced affinity for Fc.epsilon.RI on rat basophilic leukemia (RBL) cells, but the interpretation of this finding is controversial (Weetall et al., J. Immunol., 145, 3849-3854 [1990]).
Third, chimeric molecules have been constructed. Human IgE does not bind to the murine receptor (Kulczycki Jr., et al., J. Exp. Med., 139:600-616 [1974]) while rodent IgE binds to the human receptor with a reduced affinity (Conrad, et al., J. Immun., 130:327-333 [1983]); human IgG1 does not bind to IgE receptors (Weetall et al., J. Immun., 145:3849-3854 [1990]). Based on these observations, several groups have constructed human-murine chimeras or human IgE-IgG chimeras. Weetall et al., J. Immun., 145:3849-3854 (1990) made a series of human IgG1-murine IgE chimeras and concluded that the Fc.epsilon.2 and Fc.epsilon.3 domains are involved in binding murine Fc.epsilon.RI while the Fc.epsilon.4 domain is unlikely to be involved in binding to murine Fc.epsilon.RI (but may possibly be involved in binding to Fc.epsilon.RII). However, their conclusions rest primarily on lack of binding by chimeras and since three of their five chimeras lacked some interchain disulfide bonds, the conclusions drawn from these data are uncertain.
Nissim et al., EMBO J., 10:101-107 (1991) constructed a series of human-murine IgE chimeras and measured binding to RBL cells. Their study included chimeras which incorporated receptor binding into molecules which should otherwise not bind. They concluded that the entire binding site of IgE which binds with high affinity to the specialized Fc.epsilon. receptor on RBL cells could be assigned to Fc.epsilon.3.
The results reported by these authors (e.g. Helm et al., and Burt et al.,) are inconsistent. Further, in the case of anti-IgE antibodies it is difficult to eliminate the possibility of nonspecific blocking due to steric hindrance (Schwarzbaum et al., Eur. J. Immun. 19:1015-1023 [1989]). It is apparent that considerable confusion exists in the art as to the domains of IgE Fc which are involved in the binding of IgE to FCEH or in the maintenance of IgE conformation responsible for IgE binding to FCEH.
It is an object of this invention to identify polypeptides capable of differential binding to FCEL and FCEH.
It is an object herein to determine an IgE domain which is implicated in FCEH receptor binding, but which is not involved in FCEL receptor binding, and vice-versa.
It is another object herein to identify antagonists which are capable of inhibiting allergic responses, including antagonists that neutralize the FCEH or FCEL receptor-binding domains of Fc.epsilon. and immunoglobulin analogues that bind FCEL but do not bind FCEH, or that bind FCEH but not FCEL.
It is another object to provide novel polypeptides for use in the assay of Fc.epsilon. receptors and for use as immunogens or for selecting anti-IgE antibodies.